In many applications, it is desirable to detect the presence of one or more molecular structures in a sample. For example, a DNA or RNA sequence analysis is very useful in genetic and disease diagnosis, toxicology testing, genetic research, agriculture and pharmaceutical development. Likewise, cell and antibody detection is important in disease diagnosis.
A number of techniques have been developed for molecular structure detection. In DNA and RNA sequence detection, two procedures are generally used, autoradiography and optical detection. Autoradiography is performed using .sup.32 P or .sup.35 S. For DNA sequence analysis applications, nucleic acid fragments are end labeled with .sup.32 P. These end labeled fragments are separated by sizes, then exposed to x-ray film for a specified amount of time. The amount of film exposure is related directly to the amount of radioactivity adjacent to a region of film.
The use of any radioactive label is associated with several disadvantages. First, prolonged exposure to radioactive elements increases the risk of acquiring genetic diseases, such as cancer. As such, precautions must be implemented when using radioactive markers or labels to reduce the exposure to radioactivity. Typically, workers must wear a device to continually monitor radioactive exposure. In addition, pregnant females should take additional precautions to prevent the occurrence of genetic mutations in the unborn.
Further, the incorporation of a radioactive label into a nucleic sequence increases the complexity and cost of the entire sequence analysis process. Although the chemistry is commonplace, it nonetheless necessitates an additional step. The film exposure also increases the required labor and cost of materials. The increase in cost is due to the extra reagents necessary for end labelling, as well as precautionary steps necessary for the safe handling of radioactive materials.
The radioactive detection scheme has sensitivity limitations in both the temporal and spatial domains. The use of radioactive labelling currently has a spatial resolution of one millimeter. Additional hardware and software are required to reduce the spatial resolution below one millimeter. The sensitivity of detection of the autoradiographic film is related directly to the amount of time during which the radioactive labelled fragments are exposed to the film. Thus, the exposure time of the film may range from hours to days, depending upon the level of radioactivity within each detection test site. A .beta. scanner may drastically reduce the time required for film exposure during radiography. However, the use of the .beta. scanner significantly increases the expense associated with this type of detection, and has intrinsically poor spatial resolution.
Optical detection of fluorescent labelled receptors has also been utilized to detect molecular binding. Briefly, for DNA sequence analysis applications, a base-specific fluorescent dye is attached covalently to the oligonucleotide primers or to the chain terminating dideoxynucleotides. The appropriate absorption wavelength for each dye is chosen and used to excite the dye. If the absorption spectra of the dyes are close to each other, a specific wavelength can be chosen to excite the entire set of dyes.
A separate optical detection technique involves the use of a dye, for example, ethidium bromide, which stains duplexed nucleic acids. The fluorescence of these dyes exhibits an approximate 20-fold increase when it is bound to duplexed DNA or RNA, when compared to the fluorescence exhibited by unbound dye, or dye bound to single-stranded DNA. This type of dye is used to detect the presence of hybridized DNA (or RNA) during a hybridization experiment. Although the use of optical detection methods increases the throughput of the sequencing experiments, the disadvantages are serious.
The various dyes employed in using fluorescent labelled molecules have been shown to be mutagenic and, hence, carcinogenic. For example, ethidium bromide can intercalate into human DNA, and during replication, specific regions of DNA may not be replicated exactly. Proper precautions, such as gloves and careful experimental techniques, can provide the worker with protection against dye penetration. However, it is advantageous to reduce the exposure to these mutagenic agents. In addition, pregnant females should take extra precautions to prevent the occurrence of genetic mutations in the unborn.
Incorporation of a fluorescent label into a nucleic acid sequence increases the complexity and cost of the entire process. Although the chemistry is commonplace, it necessitates an additional step. The increase in cost is due to the extra reagents necessary for fluorescent labeling, as well as precautionary steps necessary for safe handling of mutagenic materials.
Furthermore, the use of multiple fluorescent dyes may affect the electrophoretic mobility of the labelled molecules. Scientists have found that a mobility correction factor is required for the sequence data because the fluorescent dyes influence the electrophoretic mobility of the nucleic fragments to which they are bound. The use of an optical detection method almost always necessitates the use of a laser with one or more lines. Extra precautions, depending upon the chosen wavelength, must be taken to ensure safe operation of the laser. Depending upon the required output power, this investment may range from a few hundred to many thousands of dollars.
Aside from the laser, additional hardware must be purchased for the operation of a fluorescent-based system. For example, a fluorescent detector and optics which transmit the fluorescent data to the detector are necessary elements of this system. If multiple excitation emission wavelengths are chosen, then a control system must be included to select the proper wavelength associated with a particular dye. Additionally, the fluorescent dyes must be purchased from a vendor.
Therefore, a need has arisen in the industry for a safe, low-cost, fast and accurate method and apparatus for detecting molecular structures at reduced complexity.